Project Summary The cornea is the most densely innervated structure within the human body. Aberrant corneal nerve function is associated with diseases such as diabetes and direct eye issues such as neurotrophic keratopathy, corneal opacities, dry eye syndrome, and keratoconus. Some neurological disorders (e.g., migraines) are correlated with abnormal corneal nerve patterning which is likely to profoundly influence corneal signaling. Understanding how corneal nerve signaling changes during normal development and during disease progression, such as type 2 diabetes, may provide insight into the etiology of eye diseases and neurological disorders in general. Also, corneal nerve damage or dysfunction can originate following a variety of surgeries, including laser in situ keratomileusis (LASIK), small incision lenticule extraction (SMILE), laser epithelial keratomileusis (LASEK), and photorefractive keratectomy (PRK). Knowledge of corneal nerve signaling is very limited at present. Before progress on understanding the role of corneal nerve function in disease states can be made, better tools need to be developed and refined. Recent studies have utilized genetically encoded Ca2+ indicators (GECIs) to allow high-fidelity Ca2+ imaging in excitable cells to help understand neural circuitry. When paired with the appropriate promoter, murine models express GECIs in specific neural subtypes or in all neurons. Our group has recently designed a GECI mouse that expresses a Ca2+ indicator (GCaMP6f) in corneal nerves which could lead to development of an imaging platform to overcome previous limitations (e.g., requires contact, inability to measure from individual cells or axons, small field of view) in recording corneal nerve signaling. Our group also has extensive experience applying infrared neuromodulation to a wide variety of tissues. In order to decode neural circuits, it is important to both measure and perturb signals from an entire network. The goal of this proposal is to establish the feasibility of using genetic indicators for assessing corneal nerve function. Although GECIs have never been used for imaging in the cornea, it is a particularly appealing target. Since the cornea is transparent, has low auto-fluorescence, and nerves are sparse in the axial direction, it is likely that very high signal to noise can be achieved and large fields of view can be interrogated. In this project, we will build a high-speed fluorescence microscope system capable of infrared neuromodulation, develop animal handling protocols (e.g., animal restraint with stereotactic system), and perform a pilot study (effect of stimuli on diabetic and control mice). If successful this approach can fill the extensive gap in technology and finally provide an exciting new platform for uncovering critical questions on how corneal nerves function under normal conditions, during the etiology of disease, and during the application of potential new drugs and therapies.